Pharmaceutical composition comprising an antigen

ABSTRACT

The inventions discloses a pharmaceutical composition comprising an antigen, an immunostimulating substance selected from neuroactive compounds, hormones, compounds having a growth hormone activity, and mixtures thereof, and a polycationic polymer.

[0001] The invention relates to a pharmaceutical composition especiallyto be used as a vaccine.

[0002] Vaccines are a very successful, yet cost saving medicalintervention. Several catastrophic illnesses including small pox andpoliomyelitis have been, due to intense vaccination programmes,eliminated from the face of this earth or are on the brink of extinction(Nossal, Nat Med 4, (1998), 475-476). In fact, vaccines can save morelives (and money) than any other medical intervention. Although thisnotion is valid for a whole panel of diseases including tuberculosis,diphteria, pertussis, measles and tetanus, there are no effectivevaccines for numerous ailments including most viral infections, such asAIDS, and other illnesses including malaria, or even cancer. Inaddition, the rapid emergence of antibiotic resistant bacteria andmicroorganisms calls for alternative treatments with vaccines being alogical choice. Finally, the great need for vaccines is also illustratedby the fact that infectious diseases, rather than cardiovasculardisorders or cancer or injuries remain the largest cause of death anddisability in the world (Bloom et al, Nat Med 4, (1998), 480-484).

[0003] The main problem in the field of vaccines is that traditionalvaccines (and/or the immune modulating compounds contained within thesepreparations) are designed to induce high levels of antibodies (Harlowet al, Cold Spring Harbor: Cold Spring Harbor Laboratory, (1988)).Unfortunately, antibodies on their own are not effective in preventingmany diseases including most illnesses caused by viruses, intracellularbacteria, or certain parasites. Examples are pathogens such as theabove-mentioned HIV virus or Plasmodium spec. in case of malaria. Inaddition, these vaccines likely will not be effective in cancer. Innumerous experimental systems it has been shown that the cellular arm ofthe immune system, including T cells, rather than the humoral arm, isimportant for these indications. Therefore, novel, innovativetechnologies to overcome the limitations of conventional vaccines areneeded. The focus must be on technologies that reliably induce thecellular immune system, including antigen specific T cells, whichrecognize molecules expressed on pathogen infected cells. Ideally,vaccines are designed that induce both T cells distinguishing diseased,and/or infected cells from normal cells and, simultaneously, antibodiessecreted by B cells recognizing pathogens in extracellular compartments.

[0004] Commonly, vaccines are administered as a combination ofpathogen-derived antigens together with compounds that induce or enhanceimmune responses against these antigens (these compounds are usuallytermed adjuvants). Examples of antigens are whole organisms such asinactivated or attenuated viruses or bacteria, fungi, protozoa or evencancer cells. Antigens may also consist of sub-fractions of thesesorganism/tissues, proteins or, in their most simple form, peptides.Antigens can also be recognized by the immune system in form ofglycosylated proteins or peptides and may also be or containpolysaccharides or lipids. Short peptides can be used since for examplecytotoxic T cells recognize antigens in form of short usually 8-11 aminoacids long peptides in conjunction with major histocompatibility complex(MHC) (Rammensee et al., Immunogenetics 41, (1995), 178-228). B cellsrecognize longer peptides starting at around 15 amino acids (Harlow etal, Cold Spring Harbor: Cold Spring Harbor Laboratory, (1988)). Bycontrast to T cell epitopes the three dimensional structure of B cellantigens may also be important for recognition by antibodies. In orderto obtain sustained, antigen-specific immune responses, adjuvants needto trigger immune cascades that involve all cells of the immune systemnecessary. Primarily, said adjuvants are acting, but are not restrictedin their mode of action, on so-called antigen presenting cells (APCs).These cells usually first encounter the antigen(s) followed bypresentation of processed or unmodified antigen to immuneeffector-cells. Intermediate cell types may also be invovled. Onlyeffector cells with the appropriate specificity are activated in aproductive immune response. The adjuvant may also locally retainantigens and co-injected other factors. In addition the adjuvant may actas a chemoattractant for other immune cells or may act locally and/orsystemically as a stimulating agent for the immune system.

[0005] Human growth hormone (HGH) is a pituitary-derived factorprimarily described for its ability to promote growth acceleration(reviewed in Neely et al, Annu Rev Med 45, (1994), 407-420). The firstpatient was treated with growth hormone obtained from pituitary extractsas early as 1958. Recombinant HGH is available for roughly 15 years nowand has been used extensively in the clinic. Side effects of recombinantHGH are rare. Efficacy of recombinant HGH preparations has beendemonstrated in a wide spectrum of diseases including Turner syndrome,idiopathic short stature, growth hormone deficiency and renal failure.

[0006] Whilst numerous studies have confirmed the growth promotingeffect of HGH, relatively few reports address a possible interaction ofthis molecule with cells of the immune system. Stephenson and Melling,who showed that HGH greatly enhances the efficacy of a viral vaccinepreparation, first demonstrated the usefulness of HGH in a vaccinecontext (Stephenson et al., J Infect Dis 164, (1991), 188-191). Theyco-injected HGH with a vaccine for tick-born encephalitis (TBE) virus,an endemic virus transmitted by ticks. In animal experiments HGHpotentiated vaccine efficacy and led to the protection of animals afteronly one injection of the vaccine. The mechanism of how HGH enhancedvaccine efficacy is unclear, but it was speculated that cell mediatedimmunity played a significant role. There is further, albeitcircumstantial, evidence that HGH may indeed induce cellular immunereactions: Mellado et al. demonstrated that if an antigen derived fromhuman immuno deficiency virus (HIV) is applied to mice, they develop aso called Th1 type T helper cell response indicative of a cellularimmune response (Mellado et al., Vaccine 16, (1998), 1111-1115). Takentogether, there is circumstantial evidence that HGH, which is viewed asexemplary for a whole class of primarily neuroactive compounds (see e.g.Levite, PNAS 95 (1998), 12544-12549, Scholzen et al, Exp. Dermetal. 7(1998), 81-96), may have a positive effect on the immune system, but themechanisms remain unclear.

[0007] Polycationic polymers, for example the polycationic amino acidpolymers poly-L-arginine and poly-L-lysine, have been shown to allowvery efficient charging of antigen presenting cells (APCs) with antigensin vitro and in vivo (Buschle et al., Gene Ther Mol Biol 1, (1998),309-321; Buschle et al., Proc Natl Acad Sci USA 94, (1997), 3256-3261;Schmidt et al., Proc Natl Acad Sci USA 94, (1997), 3262-3267). This isthought to be the key event for triggering immune cascades eventuallyleading to the induction of antigen specific immune effector cells thatare able to destroy or neutralize targets. It has been shown previouslythat a number of polycationic compounds exert effects on immune cells(Buschle et al., Gene Ther Mol Biol 1, (1998), 309-321; Buschle et al.,Proc Natl Acad Sci USA 94, (1997), 3256-3261;).

[0008] Co-injection of a mixture of poly-L-arginine or poly-L-lysinetogether with an appropriate antigen as a vaccine protect animals fromtumor growth in several animal models (Buschle et al., Gene Ther MolBiol 1, (1998), 309-321; Schmidt et al., Proc Natl Acad Sci USA 94,(1997), 3262-3267). Thus, a vaccine consisting of polycationic compoundsand antigen(s) is accepted in the art as being a very effective form oftreatment.

[0009] GB 1 290 141 discloses a vaccine containing antigenic materialwith a base-rich peptide as an adjuvant. According to this document thevaccine may consist (as antigenic material) of live or killed organisms,whole or disrupted, or of preparations of natural toxins or products ofthe organsim, or of preparations of extracts of the organisms, alone orin combination with one another. The base-rich peptide used as anadjuvant must contain at least 50% residues which have a free aminogroup, such as polylysine, polyornithine, polyarginine andpolydiaminobutyric acids. In WO 97/30721 the use of basic polyamino acidas preferred adjuvant for vaccines in combination with animmunomodulating peptide or protein (fragment) is described. The use ofa further immunostimulating substance to be used together with anantigen and such a polycationic polymer is neither disclosed nor madeobvious by these two documents.

[0010] U.S. Pat. No. 3,725,545 describes that it is possible topotentiate the antibody production ability of nucleic acids containingpreparations by adding cationically charged polymers in combination withsingle-stranded or multi-stranded nucleic acid polymers. Examples ofsuch polycationic polymers are polyornithine, lysozyme, DEAE-Dextran,histone, hexadimethrine bromide and polylysine.

[0011] WO 91/04052 also relates to DEAE-Dextran as a polycationicadjuvant in a vaccine composition. This polycationic adjuvant isincorporated into a vaccine which further comprises the antigenicsubstance (the antigen) and saponin as a further adjuvant. Of course,neither polycationic compounds nor saponin as combined adjuvants may beregarded as an immunostimulating substance within the course of thepresent invention.

[0012] It is the object of the present invention to provide apharmaceutical composition which allows an effective delivery to atarget cell especially to the cellular immune system.

[0013] This object is solved by a pharmaceutical composition comprising

[0014] an antigen,

[0015] an immunostimulating substance selected from neuroactivecompounds, hormones, compounds having a growth hormone activity andmixtures thereof, and

[0016] a polycationic polymer.

[0017] It has surprisingly turned out that the combination of theselected immunostimulating substance according to the present inventionand the polycationic polymer with an antigen leads to a synergisticimmunomodulating effect for a given antigen preparation. Indeed, itturned out that the effect of an immunostimulating substance selectedaccording to the present invention alone with the antigen has—foritself—even a lower effect than administering the antigen with thepolycationic polymer alone.

[0018] The immunostimulating effect of substances such as human growthhormone (HGH) has been reported (see also: EP 0 434 749 B1, U.S. Pat.Nos. 4,837,202, 5,830,877 and 5,583,109). Indeed, many of theneuroactive substances, such as pituitary growth hormones also exhibit Tcell inducing activity or cytokine secretion altering activities. Thesecompounds also may act on APCs.

[0019] The immunostimulating effect is especially given as thesesubstances e.g. induce T cells, B cells, NK cells or APCs or alter thecytokine secretion of T cells, B cells, NK cells or APCs.

[0020] It turned out, however, that administration of an antigentogether with such immunostimulating substances alone does not lead toan efficient cellular immune response although such substances mayhave—as stated above—T cell inducing activity, are able to alter thecytokine secretion of T cells (s. Levite (1998), and Scholzen et al(1998)) or activate the immune system in a more general way.

[0021] The present invention is based on the careful selection ofimmunostimulating substances to be used in connection with thepolycationic polymer. The polycationic polymer serves as adjuvant in thepresent invention. It is therefore clear that other adjuvants describedin the prior art may not be regarded as immunostimulating substances inthe course of the present invention. Although such additional adjuvantsmay be also added to the present vaccine, they cannot substitute theimmunostimulating substance selected according to the present invention.

[0022] HGH is specifically preferred for the present invention asimmunostimulating substance. Several isoforms are known for thisprotein, recombinant forms of such isoforms being specifically preferredfor the present invention. Preferably, HGH is administered in astabilized composition according to the present invention, especiallywith stabilizers such as glycine, tensides, mannitol orpolyoxyethylene-polyoxypropylene block copolymers, and buffers, such assodium phosphate or citrate (WO 89/09614, EP 0 211 601 A, EP 0 587 958A).

[0023] The antigens to be used in the present compositions are notcritical. Preferably proteins or peptides derived from a viral or abacterial pathogen or from fungi or parasites are used as such antigens(including derivatized antigens like glycosylated, lipidated,glycolipidated or hydroxylated antigens. Furthermore, carbohydrates,lipids or glycolipids may be used as antigens themselves. Preferredpathogens are selected from HIV, HBV, HCV, Influenza virus, Rotavirus,Staphylococcus aureus, Chlamydia pneumoniae, Mycobacterium tuberculosis,Streptococcus pneumoniae, Bacillus anthracis, Vibrio cholerae,Plasmodium sp. (Pl. falciparum, Pl. vivax, etc.), Aspergillus sp. orCandida albicans. The derivation process may include the purification ofa specific protein from the pathogen, the inactivation of the pathogenas well as the proteolytic or chemical derivatization or stabilizationof such a protein. In the same way also tumor antigens (cancer vaccines)or autoimmune antigens may be used in the pharmaceutical compositionaccording to the present invention. With such compositions a tumorvaccination or a treatment for autoimmume diseases may be performed.

[0024] Preferably antigens are used which induce an antigen-spcifictype-1 like tumoral and cell mediated immune response in vivo. CD4- andCD8-epitopes are preferred, especially in connection with vaccinesagainst pathogens and tumors.

[0025] Preferred immunostimulating substances include pituitary growthhormones or derivatives thereof, especially proteolytically orrecombinantly produced derivatives which exhibit the functionalproperties of the growth hormone (described e.g. in the U.S. Pat. Nos.5,854,026 or 5,849,535, 5,424,199 or 5,580,723). It has been shown thatsuch substances are also able to alter the cytokine secretion of Tcells, Bcells or NK cells, but they may also exert effects on APCs or Bcells or NK cells. Their immunostimulating effect was connected in theliterature with sepcific receptors on T cells (s. Levite (1998)).

[0026] Other preferred neuroactive compounds are selected from but arenot restricted to growth hormones, especially human growth hormone,neurokinin A, vasoactive intestinal peptide, neuropeptide Y, substanceP, Thyrotrophin (TSH), Insulin-like growth factor I (IGF-1), prolactin,lactogen, luteinizing hormone, follicle stimulating hormone,dehydroepiandosterone (DHEA), Thymosin, Thymulin, Kentsin, melatonin,semaphorins (s. Levite (19913); Scholzen et al (1998); Aronin et al,Ann. Rev. Physiol. 48 (1986), 537-549; Berczi, Acta Paediatr Suppl. 423(1997), 70-75; Chappel, J. Acq. Imm. Def. Synd. 20(5)(1999), 423-431;Goldman et al., Ann N.Y. Acad. Sci 419 (1983), 143-155; Spriggs, Curr.Op. Immunol. 11 (1999), 387-391; Delneste et al., J. Immunol. 163(1999), 3071-3075). Also functional derivatives of such compounds maypreferably be used in the present invention. A suitable method forproviding such derivatives of naturally occuring substances is disclosedin the U.S. Pat. No. 5,580,723.

[0027] It is clear to the man skilled in the art that the terms“neuroactive compounds”, “hormones”, “compounds having a growth hormoneactivity” are overlapping and many known substances fell into two oreven all three groups. However, these terms are to be understoodaccording to their meaning as generally used in the scientificliterature, especially in the literature which deals with immunologicalresearch to these substance classes.

[0028] The polycationic compound to be used according to the presentinvention may be any polycationic compound which shows thecharacteristic effect according to the WO 97/30721. Preferredpolycationic compounds are selected from basic polypeptides, organicpolycations, basic polyaminoacids or mixtures thereof. Thesepolyaminoacids should have a chain length of at least 4 amino acidresidues (see: Tuftsin as described in Goldman et al (1983)). Especiallypreferred are substances like polylysine, polyarginine and polypeptidescontaining more than 50% of basic amino acids in a range of more than 8,especially more than 20, amino acid residues or mixtures thereof.

[0029] These polycationic compounds may be produced chemically orrecombinantly or may be derived from natural sources. Preferredpolycationic compounds derived from natural sources include HIV-REV orHIV-TAT derived cationic peptides, antennapedia peptides, chitosan (orother derivatives of chitin) and other peptides derived from thesepeptides or proteins by biochemical or recombinant production.

[0030] It was very surprising that with the pharmaceutical compositionaccording to the present invention the immunostimulating effect wassignificantly higher than it could be expected from the addition of theeffects of each single component or even the addition of the effects ofthe polycation with the antigen and the immunostimulating substanceselected according to the present invention with the antigen. Moreover,it turned out that the effect of the selected immunostimulatingsubstances alone is not very high when an antigen is directly appliedwith this substance. This is true in particular if the compounds are notrepeatedly administered.

[0031] According to another aspect the present invention also relates tovaccines which comprise a composition according to the presentinvention.

[0032] Moreover, the present invention is also drawn to the use of thecomposition according to the present invention for manufacturing avaccine.

[0033] The relative amounts of the ingredients of the presentcomposition are highly depending on the necessities of the individualcomposition, e.g. the polycationic polymer to be used. In the case ofpoly-L-arginine and poly-L-lysine, preferred amounts ofantigen/immunostimulating compound/polycation lie in the range of1-10000 μg antigen per vaccination, 0.001 to 1000 units ofimmunostimulating compound per dose, especially in the case of hormones,such as HGH, and 0,1 to 1000 μg polycation.

[0034] The present compositions may be applied to a patient, e.g. avaccination candidate, in effient amounts e.g. by weekly, bi-weekly ormounthly intervals. Patients to be treated with the present compositionsmay also be vaccinated repeatedly or only once. A preferred use of thepresent invention is the active immunisation, especially of humans oraninmals without protection against the specific antigen.

[0035] The route of application for the present composition is notcritical, e.g. subcutaneous, intramuscular, intradermal or transdermalinjection is suitable as well as oral uptake.

[0036] It is also possible to apply the present composition separatedlye.g. by injecting the immunostimulating substance separatedly from theantigen/polycation composition. The present invention is therefore alsodirected to a kit comprising a composition containing the antigen andthe polycationic polymer as one component and a composition containingthe immunostimulating or chemotactic substance as a second component.

[0037] The components may be applied at the same site or time, however,an application at different sites or at a different time or for adifferent time period is also possible. It is also possible to vary thesystemic or local applications of the composition or the components,respectively.

[0038] In a further aspect the present application relates to a vaccineagainst HCV infections comprising a polycationic polymer, animmunostimulating substance and an HCV antigen being selected from thegroup consisting of Core 23-44 (KFPGGGQIVGGVYLLPRRGPRL), CORE 131-150(ADLMGYIPLVGAPLGGAARA), CORE 132-140 (DLMGYIPAV), NS3 1073-1081(CINGVCWTV), NS3 1207-1226 (SPVFTDNSSPPAVPQSFQVA), NS3 1248-1261(GYKVLVLNPSVAAT), NS3 1585-1604 (YLVAYQATVCARAQAPPPSW), NS4 1655-1675(VVTSTWVLVGGVLAALAAYCL), NS4 1765-1784 (MWNFISGIQYLAGLSTLPGN), NS41909-1929 (GEGAVQWMNRLIAFASRGNHV), NS4 1785-1804 (PAIASLMAFTAAVTSPLTTG),NS4 1787-1799 (IASLMAFTAAVTS), NS4 1805-1824 (QTLLFNILGGWVAAQLAAPG) NS41809-1821 (FNILGGWVAAQLA), and CD4- and CD8-epitopes with at least 6,preferably 8 to 10, amino acid long fragments of said antigens or strainvariants of those antigens. Numbering of the amino acid residues relatesto standard HCV nomenclature. The CD8 epitopes are especially selectedfrom Core 31-40, Core 35-44, Core 132-140, NS3 1585-1593, NS3 1585-1594,NS4 1666-1675, NS4 1769-1777, NS4 1773-1783, NS4 1787-1795, NS41789-1797, NS4 1789-1798, NS4 1807-1816, NS4 1809-1817 und NS41811-1820.

[0039] These HCV epitopes have been shown to be surprisingly effectivein vaccines containing polycationic peptides, especially polyarginine.

[0040] These carefully selected peptides are conserved in the dominantgenotypes of HCV which are—especially in combination withpolyarginine—highly immunogenic and therefore surprisingly effective asvaccines. Although a vast variety of peptides and epitopes have beendescribed as potential vaccine candidates for HCV it turned out thatonly the above mentioned epitopes together with polycationic substances(especially polyamino acids) such as polyarginine could provide thedesired results. Therefore, the present invention also relates to avaccine comprising the selected HCV antigens together with the polyaminoacid adjuvants, especially polyarginine, and optionally the immunogeniccompounds according to the present invention.

[0041] Details of the present invention are described by the followingexamples and the figure but the invention is of course not limitedthereto.

[0042]FIG. 1 shows the synergistic action of poly-L-arginine and humangrowth hormone in the induction of antigen specific T cells.

[0043]FIG. 2 shows the synergistic action of poly-L-arginine andsubstance P in the induction of antigen specific T cells.

EXAMPLES Example 1 Induction of Antigen Specific T Cells is GreatlyEnhanced by Coinjection of Combination of poly-L-arginine and HumanGrowth Hormone

[0044] Mice C57BL/6 (Harlan/Olac) Peptide VYDFFVWL derived from mousetyrosinase related protein-2. Restricted to H-2Kb (Bloom et al., 1997)Dose: 100 μg/mouse. Control peptide SIINFEKL derived from ovalbumin.Restricted to H-2Kb (Carbone and Bevan. 1989). Poly-L-arginine 60 (pR60)poly-L-arginine with an average degree of polymerization of 60 arginineresidues; SIGMA chemicals Dose: 100 μg/mouse Human Growth Hormone (HGH)0,02 IU/injection SAIZEN, Laboratoires Serono)

[0045] Peptides were synthesized by standard solid phase F-mocsynthesis, HPLC purified and analysed by mass spectroscopy for purity.

[0046] Experimental Groups (5 Mice Each)

[0047] 1) TRP-2 peptide

[0048] 2) TRP-2 peptide+HGH

[0049] 3) TRP-2 peptide+pR 60

[0050] 4) TRP-2 peptide+pR 60+HGH

[0051] On day 0 mice were injected subcutaneously with a total volume of100 μl containing the above mentioned compounds. Animals were sacrificed10 days after injection of the vaccine and mesenteric and inguinal lymphnodes harvested. Results are illustrated in FIG. 1.

[0052] Lymphocytes were prepared from lymph nodes as follows: cells werepassed through a 70 μm sieve and washed twice with DMEM medium (GIBCOBRL) containing 2,5% fetal calf serum (FCS; SIGMA chemicals). Cells wereadjusted to 10⁷ cells/ml in complete medium (DMEM +10% FCS).IFN-γ-ELISPOT assays were carried out in triplicate as described(Miyahira et al., 1995). This method is a widely used procedure allowingthe quantification of antigen-specific T cells. Lymphocytes wererestimulated with TRP-2 peptide or with an ovalbumin-derived peptide(SIINFEKL) with the same MHC restriction serving as negative control.

[0053] Spots representing single T cells specific for the peptide usedfor re-stimulation (and immunization) were counted. The number ofbackground spots observed with cells incubated without peptide(s) wassubtracted from all samples. There were no spots detected when theovalbumine derived peptide was used. The number of spots resulting fromthe restimulation with the TRP-2 derived petide are shown below for eachgroup of mice.

Example 2 Preferred Antigens to be Used for Providing a VaccineComposition According to the Present Invention

[0054] 1. HCV: antigens according to table 1 and the antigens disclosedin Lamonaca et al., Hepatology 30(4) (1999), 1088-1098.

[0055] Hepatitis C Peptides TABLE 1 CD4 epitopes Sequence References CD8epitopes Sequences References Core 23-44 KFPGGGQIVGGVYLLPRRGPRL(Hoffmann et al., 1995) Core 132-140 DLMGYIPAV (Sarobe et al., 1998)E2/NS1 — — E2/NS1 723-731 FLLLADARV (Wentworth et al., 1996) NS31248-1261 GYKVLVLNPSVAAT (Diepolder et al., 1997) NS3 1073-1081CINGVCWTV (He et al., 1999; Rehermann et al., 1996) Core 131-151ADLMGYIPLVGAPLGGAARA (Hoffmann et al., 1995)

[0056] 2. HIV: antigens according to table 2 TABLE 2 Peptide SequenceGag 77-85 SLYNTVATL Envelope 77-85 DPNPQEVVL POL 476-484 ILKEPVHGV

[0057] 3. Epstein-Barr virus: the antigens according to table 1 ofRickinson et al Ann. Rev. Immunol. 15 (1997), 405-431.

Example 3 Co-Injection of Substance P and pArg

[0058] Substance P was tested as a further example for a neuroactivepeptide (Marx, Science 205 (1979), 886). Substance P (RPKPQQFFGLM-NH₂;SP) was synthesized and purified according to standard procedures.Experiments were conducted as in Example 1 with the exception thatspleens instead of lymph nodes were used.

[0059] Splenocytes werde prepared from spleens as follows: cells werepassed through a 70 μm sieve and washed once with DMEM medium (GIBCOBRL). Red blood cells were lysed with “red blood cells lysis buffer”(Sigma) for 1 minute and washed twice with DMEM medium (GIBCO BRL).Cells were adjusted to 3*10⁶ cells/ml in complete medium (DEMEM+5% FCS).IFN-γ-ELISPOT was carried out as described (Miyahira et al., 1995).

[0060] Experimental Groups (2Mice Each)

[0061] 1) TRP-2 peptide

[0062] 2) TRP-2 peptide+pR 60

[0063] 3) TRP-2 peptide+pR 60+substance P

[0064] Per injection 10 nmoles SP have been administered to mice.

[0065] The results are depicted in FIG. 2. It is demonstrated with thepresent example that co-injection of SP and poly-L-arginine results ingreatly enhanced generation of T cells in an IFN-γ-ELIS-POT essay.

1. Pharmaceutical composition comprising an antigen, animmunostimulating substance selected from neuroactive compounds,hormones, compounds having a growth hormone activity, and mixturesthereof, and a polycationic polymer.
 2. Composition according to claim1, characterized in that the antigen is a protein derived from a viral,parasitic or bacterial pathogen.
 3. Composition according to claim 1,characterized in that the antigen is a tumor antigen.
 4. Compositionaccording to claim 1, characterized in that the antigen is an autoimmuneantigen.
 5. Composition according to any one of the claims 1 to 4,characterized in that the polycationic compound is a basic polypeptide,an organic polycation, a basic polyaminoacid or mixtures thereof. 6.Composition according to any one of claims 1 to 5, characterized in thatthe polycationic compound is polylysine, polyarginine, a polypeptidecontaining more than 50% of basic amino acids in a range of more than 8,especially more than 20, amino acid residues or mixtures thereof. 7.Composition according to any one of claims 1 to 6, characterized in thatthe polycationic compound is derived from the REV-protein or theTAT-protein of HIV, chitosan or other chitin derivatives.
 8. Compositionaccording to any one of claims 1 to 7, characterized in that theneuroactive compounds are selected from growth hormones, especiallyhuman growth hormone, neuro-kinin A, vasoactive intestinal peptide,neuropeptide Y, substance P, Thyrotrophin (TSH), Insulin-like growthfactor I (IGF-1), prolactin, lactogen, luteinizing hormone, folliclestimulating hormone, dehydroepiandrosterone (DHEA), Thymosin, Thymulin,Kentsin, melatonin, semaphorins.
 9. Vaccine, comprising a compositionaccording to any one of claims 1 to
 8. 10. Use of a compositionaccording to any one of claims 1 to 8 for manufacturing a vaccine. 11.Kit comprising a component containing an immunostimulating substanceselected from neuroactive compounds, hormones, compounds having a growthhormone activity, and mixtures thereof, and a component containing apolycationic polymer and an antigen.
 12. Vaccine, comprising a HCVantigen selected from the group of consisting of Core 23-44(KFPGGGQIVGGVYLLPRRGPRL), CORE 131-150 (ADLMGYIPLVGAPLGGAARA), CORE132-140 (DLMGYIPAV), NS3 1073-1081 (CINGVCWTV), NS3 1207-1226(SPVFTDNSSPPAVPQSFQVA), NS3 1248-1261 (GYKVLVLNPSVAAT), NS3 1585-1604(YLVAYQATVCARAQAPPPSW), NS4 1655-1675 (WTSTWVLVGGVLAALAAYCL), NS41765-1784 (MWNFISGIQYLAGLSTLPGN), NS4 1909-1929 (GEGAVQWMNRLIAFASRGNHV),NS4 1785-1804 (PAIASLMAFTAAVTSPLTTG), NS4 1787-1799 (IASLMAFTAAVTS), NS41805-1824 (QTLLFNILGGWVAAQLAAPG) NS4 1809-1821 (FNILGGWVAAQLA), and CD4-and CD8-epitopes with at least 6, preferably 8 to 10, amino acid longfragments of said antigens or strain variants of said antigens and apolycationic polymer.
 13. Vaccine according to claim 12, wherein thefragments are selected from Core 31-40, Core 35-44, Core 132-140, NS31585-1593, NS3 1585-1594, NS4 1666-1675, NS4 1769-1777, NS4 1773-1783,NS4 1787-1795, NS4 1789-1797, NS4 1789-1798, NS4 1807-1816, NS41809-1817 und NS4 1811-1820.
 14. Vaccine according to claim 12 or 13,wherein the polycationic polymer is a basic polypeptide, preferably apolyamino acid, especially polyarginine.
 15. Vaccine according to any ofclaims 12 to 14 further comprising an immunostimulating substanceselected from neuroactive compounds, hormones, compounds having a growthhormone activity and mixtures thereof.